Method for measuring bubble H + flow velocity by non-damage micro-measurement technique

The traditional method for measuring H + transport in the vacuole membrane is accomplished by pH fluorescent dyes in vitro. However, the high-purity bubble membrane separation in this method is complicated, and the sensitivity to the weak H + real-time monitoring is not enough, which has caused the researchers to use it. In recent years, non-damage micro-measurement technology (NMT) has been widely used in electrophysiology research. NMT has the advantages of non-damage, high sensitivity and real-time monitoring of the entire process. So far, although the Ca2 + flow of the beet vacuole has been measured by NMT, there has been no report on the measurement of the vacuole H + flow rate. Therefore, the establishment of a method for measuring the bubble and the H + flow rate of the cell membrane system is of great significance for the study of proton movement and membrane protein activity.

In 2011, the research team of Li Yinxin of the Institute of Botany, Chinese Academy of Sciences made several amendments to the method of non-damaging micro-measurement (NMT) to measure the bubble H + flow, so that NMT can measure the bubble H + flow more accurately. At the same time, the newly established system was used to determine the difference in vacuolar H + flow rate of transgenic tobacco TS15 and WT tobacco under NaCl stress, and the activity of H-ATPase and PPase on the vacuole membrane was measured by NMT method. The research first simplifies the method of liquid bubble extraction based on the characteristics of NMT's visual measurement and omits the cumbersome ultracentrifugation step in traditional detection. Under 200 mM NaCl stress, H + outflow increased, but TS15 H + outflow was significantly stronger than WT outflow, indicating that NHX1 had higher activity in transgenic plants. When ATP and PPi were applied under 200mM NaCl, the change of vacuole â–³ H + flow rate reflected the activity of H-ATPase and PPase; research showed that after applying ATP, the transgenic tobacco vacuole showed a greater â–³ H + flow rate; The plants had higher V-ATPase activity than WT plants at 200 mM NaCl. At the same time, using traditional methods to calculate H-ATPase and PPase enzyme activity results also confirmed the accuracy of NMT determination. Compared with the traditional fluorescence method, NMT has a greater advantage in the operation process, the accuracy of the results and the amount of information on the conclusions.

This study establishes and revises a suitable and accurate method for measuring bubble H + flow rate. The difference in ion current can also reflect more physiological characteristics, and it is a new method that can replace traditional V-ATPase and PPase activity measurement methods. This simple method will enable the detection and analysis of subcellular and organelle level ion flow rates in the future.